morphology and function of malpighian tubules and associated structures in the cockroach,...

7/25/2019 Morphology and Function of Malpighian Tubules and Associated Structures in the Cockroach, Periplaneta Americana

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Morphology and Function of Malpighian Tubules and

Associated Structures in the Cockroach,

Periplaneta americana

BETTY

J.

WALL, JAMES L. OSCHMAN AND BARBARA A. SCHMIDT

Depar tment

of

Bi o l oy i c u l Sciences, Northwestern University,

Eua nst on, Illinois 60201

A B S T R A C T

This p aper describes the different regions of the Malpighian

tubules and the associated structures (ampulla, midgut, ileum) in the cock-

roach, Periplaneta americana. There are about 150 tubules in each insect.

Each tubule consists of a t least three parts. T he sho rt distal region

is

thinner

than the other parts and is highly contractile. The middle region comprises

most of t he tubule len gth and is composed of primary an d stellate cells. Pri-

mary cells contain numerous refractile mineral concretions, while stellate cells

have smaller nuclei, fewer organelles, simpler brush border, and numerous

multivesicular bodies. Symbiont protozoa are sometim es prese nt within the

lume n of t he middle region nea r where it opens into the proximal region of th e

tubule. The latter is a short region that drains the tubular fluid into one of

the six ampullae. These are contractile diverticula of the intes tine located a t

the midgut-hindgut junction. The ampulla

is

highly contractile, and consists

of a layer of epith elial cells surroun ding a cavity that opens in to the gut via

a narrow slit lined by cells

of

unusual morphology. The proximal region

of

the

tubule and the ampulla resemble the midgut in that they have similar micro-

villi, basal infolds, and distribution of mitochondria. This suggests an endoder-

ma1 origin and reabsorptive function for the proximal region of the tubule and

for the ampulla.

A

number of inclusions found within the tubule cells are

described, including peroxisomes and modified mitochondria. Current theories

of fluid transp ort ar e evaluated with re gard to physiological and morphological

cha rac ter ist ics of Malp ighian tubules. The possible role of long narrow chan -

nels such a s those between microvilli and within basal folds is considered, as

is th e mechanism by which these structures are formed and maintained. Also

discussed i s the role of peroxisomes and sy mbionts in the excretory process.

Osmoregulation and excretion in insects

occur in two steps. First, a primary secre-

tion or urine is produced by the Malpighian

tubules. This fluid, which resembles in

many ways a filtrate

of

the blood (Ram-

say, '58; Farquharson, '74; Maddrell and

Gardiner, '74) flows into the gut and ac-

cumulates in the rectum, where the sec-

ond, reabsorptive phase takes place (re-

viewed by Phillips, '70; Maddrell, '71 ; Wall

and Oschman, '75). Here substances that

are required for the metabolism of the

animal are reabsorbed into the blood while

wastes are retained in the rectal lumen,

concentrated, and excreted. Hence the

Malpighian tubule-rectum system corre-

sponds functionally to the nephridia of

Annelida and Onychophora and to the kid-

ney of vertebrates.

J.

MORPH.,146: 265 306.

Current interest in Malpighian tubule

structure and function was stimulated by

the studies of Wigglesworth ('3la,b,c) and,

more recently, of Ramsay ('52, '53, '54,

'55a,b, '56, '58, '61) who devised methods

for isolating individual tubules in vitro

and collecting the secreted fluid. Berridge

('66) devised a defined medium in which

the tubules could secrete for very long

periods. Detailed information was then ob-

tained on the composition of the secreted

fluid and the effects of bathing medium

composition on rate of formation and com-

position of the secreted fluid (Berridge,

'68, '69). These studies have contributed

to the development of our current theories

on the mechanism by which various epi-

thelia form fluid secretions (reviewed by

Oschman and Berridge, '71; Berridge and

265


2/42

266

B .

J . W A L L , J . L . O S C H M A N A N D

B .

A . S C H M I D T

Oschman, '72; Oschman et al., '74). It is

now thought that movement of the aque-

ous component of secretions may be the

consequence of an osmotic gradient estab-

lished by pumping some solute into a con-

fined space or infolding of the cell surface.

It was first proposed that intercellular

spaces might be the sites of the osmotic

gradients responsible for water absorption

in the gall bladder (Kaye et al., '66; Dia-

mond and Tormey, '66a,b). Extension of

the model to foldings of the cell surface

such as microvilli and basal infoldings

was suggested by Diamond and Bossert

('68) and applied to Malpighian tubules

by Berridge and Oschman ('69). Although

this

is

not the only theory of fluid secre-

tion (see

DISCUSSION ,

i t

does achieve an

integration of physiological findings with

ultrastructural features of the transport-

ing cells.

The morphology of Malpighian tubules

has been described for a number of in-

sect species (Baccetti et al ., '63; Beams

et al.,

'55;

Berkaloff, '61; Berridge and

Oschman, '69; Bradfield, '53; Byers, '71;

Eichelberg and Wessing, '75; Fuller, '66;

Grinyer and Musgrave, '64; Jarial and

Scudder, '70; Kessel, '70; Mazzi and Bac-

cetti, '63; Messier and Sandborn, '66;

Meyer, '57; Smith and Littau, '60; Sohal,

'74; Taylor, '71a,b, '73; Tsubo and Brandt,

62;

Wessing, '65; Wessing and Eichel-

berg, '69a,b; Wigglesworth and Salpeter,

'62). These studies have, however, pro-

vided little information on the region

where the tubules drain into the gut. Also,

with a few exceptions, there has been little

detailed information on tubules that are

differentiated into several regions. The

purpose of this paper is to describe in

detail the regional specializations along

the pathway of urine flow that may be

indicative of different functional capaci-

ties such as secretion, reabsorption, and

storage of metabolites. We also present

some measurements of the tubular fluid

composition and discuss the mechanism

of secretion. Finally, some of the informa-

tion obtained from this study provides

clues about the embryological origin of

the Malpighian tubules as well as the man-

ner in which foldings of the cell surface

are formed and maintained.

M A T E R I A L S A N D METHODS

Animals. Adult male Periplaneta amer-

icana were used in this study. Stock cul-

tures were maintained in 12 hours light

with water and food (oatmeal or Lab Chow)

continuously available.

Physiology. To determine the concen-

tration of the fluid secreted by cockroach

Malpighian tubules, animals were anes-

thetized with COz and then dissected open

under Ringer solution. The tubules were

dissected free and removed into a sepa-

rate drop of Ringer solution under Paraf-

fin oil (e.g. Berridge, '66). The Ringer

solution was the same as that used by

Treherne ('61). The secreted fluid as well

as a sample of the Ringer solution were

analyzed for freezing point depression

using the method of Ramsay and Brown

Morphology. Malpighian tubules and

associated structures (ampulla, midgut,

ileum) were fixed in 2.5% glutaraldehyde

with

0.05 M

phosphate buffer and

5%

su-

crose, pH 7.2 to 7.4. Tissues were postfixed

in osmium in block in aqueous uranyl

acetate. They were then dehydrated in

ethanol, embedded in Araldite, and sec-

tioned with the Huxley microtome. Sec-

tions were stained with lead citrate and

uranyl acetate. Micrographs were taken

with either the RCA-EMU-3F or the

Hi-

tachi HU -l l-E. To study regional changes

in morphology along the length of the

tubule, one tubule was divided into five

parts that were embedded and sectioned

separately.

Uranium-calcium staining. A method

for tracing intercellular spaces has been

developed in which fixation and uranium

in

block staining are done in the presence

of calcium ions. The rationale was that

extracellular polymers might be present

that are cross-linked with calcium ions,

and that these substances might be re-

tained in the tissue

if

calcium was present

in all of the solutions used to process the

specimens. Although the method worked

(fig. 22) further study will be required

to determine if the mechanism of staining

involves replacement of bound calcium

ions with uranium, or

if

calcium is acting

as a mordant for binding of colloidal ura-

nium. The method consisted of fixing the

tissue in

2.5%

glutaraldehyde buffered

in 0.1 M s-collidine with

0.005 M

CaCh

and 0.005

M

KCl added. The tissue was

then processed through wash and osmium

solutions that were buffered in the same

('55).


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MALPIGHIAN TUBULE STRUCTURE AND FUNCTION

267

way as the fixative, and with CaClz and

KC1 present. Finally, the tissue was stained

for two hours in 0.5% aqueous uranium

acetate plus

0.005 M

CaC12 and KC1. The

tissues were then dehydrated and embed-

ded in Spurr resin.

L a n t h a n u m t r e a t m e n t .

Additional in-

formation on the morphology of extracel-

lular channels was obtained by fixing tu-

bules in 2.5% glutaraldehyde in 0.1 M

s-collidine buffer containing 5 sucrose

and 0.005 M each of CaCl2, LaCL, and

MgC12. These tissues were then processed

without osmium treatment and sections

were examined without further staining.

Stellate cells . A variety of staining

methods were used in attempts to locate

stellate cells

in

cockroach tubules. The

method that proved most reliable was to

dissect the tubules in Ringer solution,

place them on albumen-coated slides, fix

in Carnoys solution, wash in distilled wa-

ter, and stain in 0.01% aqueous toluidine

blue for 20 minutes. The specimens

were

then dehydrated

in

ethanol and mounted

in

Permount.

RESULTS

General descr ip t ion

The general arrangement of the Mal-

pighian tubules and associated structures

is illustrated in figure 1. There are 144-

192 tubules in this insect (Meyers and

Miller, 69; Crowder and Shankland, 72)

although figure 1 shows only one tubule

in its entirety. Each tubule is about 2-

3

cm long and 4 0 4 0 p thick. The tubules

extend throughout the abdomen, and are

held

in

intimate contact with the fat body

and intestine by fine tracheae. The tubules

are highly contractile, owing to muscles

that extend in a spiral fashion along their

length. The contractility of the tubules

has been noted for some time (e.g. Leger

and Duboscq, 1899) and

it

appears that

true muscles are present in conjunction

with some but not all of the contractile

tubules (reviewed by Snodgrass,

35).

The

structure of the musculature of the tu-

bules has been described previously (Crow-

der and Shankland, 72) and will not be

elaborated upon here. The tubules con-

tract vigorously

in

animals that have been

dissected open under Ringer solution, al-

though there is much variation in the rate

of contraction.

We have identified three regions in these

xirnal

Ion

idgut

trachea

Fig.

1

General arrangemen t of th e structures

described in th is paper. T he full length of only one

of the Malpighian tubules is shown. The mid dle

region is th e longest part. T he short thin dis tal

region is highly contractile and the short proxi-

ma l region inserts into the am pulla. Each of th e

six ampullae drains about

24-32

tubules. The am-

pullae are contractile enlargements on the intes-

tinal surface at the juncti on between midgut and

ileum. Trach eae branch over the midg ut surface

an d send processes into the am pullae. Fine tra-

cbeoles also attach the tubules to other organs

such as fat body.

tubules, distal, middle, and proximal. The

route of flow is from distal region to proxi-

mal region. The middle region is longest,

and further study could reveal that it is

divided into smaller sections. The clear

distal region of the tubule is much shorter

(about 0.8 mm or

3

of the total length)

and is thinner (about

30 p

than the

middle region of the tubule. Movements

of the distal region of the tubule are not

correlated with those of the middle region,

and are of a different sort. This region

exhibits rapid bending motions followed

by rapid return to its original position.

Contraction of the middle region results

in coiling of the tubules. The coiling can

be loose or tight, depending on the strength

of the contraction. The middle region is

variable in color. In some animals

it

is

completely yellow, while in others it is

white (also Meyers and Miller, 69). The

yellow color is probably the result of stor-

age of some compound, possibly riboflavin

(Metcalf,

43)

within the cells. Gersch

(42) divided the middle region into two

parts on the basis of the number of se-

cretion vacuoles seen in living tubules,

although there was no sharp boundary


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MALPIGHIAN TUBULE STRUCT URE AND FUNCTION

269

Fig.

2

Survey view of the short distal region. There is a single strip of muscle (m)

embed^

ded in the connective tissue. Th e muscle is capable of shor t fast contractions. Distal region

differs from other portions in th at cells lack vacuoles; lume n i s narro w, a nd m any of the long

microvilli contain extensions of filam entou s mitochondria.

x 4,600.

ance. We

do

not illustrate the crystalline

arrangement of the core as it has been

adequately described in other tissues (re-

viewed by Tandler and Hoppel, '72)

in-

cluding the fat body of the American cock-

roach (Gharagozlov,

'69)

and of the fruit

fly

(Takahashi et al.,

70),

and the mam-

malian kidney tubule (Suzuki and Mostolfi,

'67;

Youson and McMillan, '70). There

are also round membrane-bound organelles

similar in size to mitochondria but with

a homogeneous granular interior (fig.

3) .

These may be microbodies. Other organ-

elles include Golgi complexes, bits of

smooth and rough endoplasmic reticulum,

and small vesicles. Two sorts

of

microvilli

project into the lumen in the distal region,

those containing extensions of mitochon-

dria, and others containing extensions of

the smooth endoplasmic reticulum. The


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Fig.

5

Survey micrograph

of

middle region. Lumen i s larger tha n in distal region and

cells contain nu me rou s clear vacuoles an d mineralized concretions. Large dense structures

1)

resemble lysosomes observed in other insect tissues (e.g. Locke and Collins,

'65).

Penetra-

tion of mitochondria into microvilli i s variable, i.e. compar e bru sh border a t top and bottom

of picture w ith tha t on either side. Connective tissue (ct) is thick an d mu scle (m ) is embedded

in it. Mitochondria present throughout cytoplasm. Portions of blood cells show n at u pper left.

X 4,000.

271


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272

B .

J. WALL, J. L. OSCHMAN AND B. A . SCHMIDT

Fig.

6

High magnification

of

basal portion. middle region

of

tubule. Basement membrane

consists of inner granular layer (gl) and fibrillar layer (0. Connective tissue is comprised of

collagen fibrils (col) and outer granular layer. Profiles of microtubules are observed in some

of

th e interdigitating cell processes (arrows).

X

80,000.

cellular space. Thus the arrangement of

lateral membranes is similar to that of

many other tubular epithelia

in

inverte-

brates.

M i d d l e region

Figure

5

provides a survey view

of

the

middle region of the tubule, which com-

prises most of the tubule length.

A s

men-

tioned above, this region is either com-

pletely or partly yellow in freshly dissected

specimens. The lumen is larger than that

of the distal region, and the secretory cells

are larger, apparently because they are

swollen with various sorts of vacuoles.

These will be described in more detail be-

low. The cells interdigitate with each other

and are joined by septate junctions as

in

the distal region of the tubule. The mid-

dle region is comprised of two cell types,

the primary or secretory cells, and stellate

cells. The primary cells are more abun-

dant and are described first. The following

description proceeds from basal to apical

surface.

Fig.

7

Basal portion, m iddle region

of

tubule.

Cells interd igitat e extensively. Basement m emb rane

is laminated. Note profiles of microtubules

a-

rows) and mitochondria (m) in cytoplasmic inter-

digitations. Profiles

of

rough and smooth endo-

plasmic reticulum (rer and ser) are present within

th e interdigitations.

X 40,000.

Fig.

8

Basal portion

of

tubule cell, middle re-

gion, fixed i n

glutaraldehyde-lanthanum.

Unstained

section. Lanthanum is deposited within basement

mem brane (bm) an d extracellular ch anne ls be-

tween interdigitations. X 52,000.


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M A L P IG H I AN T U B U L E S T R U C TU R E A N D F U N C T I O N

2

73


10/42

274 B . J.

WALL.

J .

L. OSCHMAN AND

B . A .

SCHMIDT

Basal surface

Figure

6

reveals details of the tubule-

hemolymph interface. The outermost layer

consists of fine granules adhering to the

collagen fibers. This connective tissue

sheath becomes much thicker toward the

proximal region of the tubule. The colla-

gen fibers appear to be embedded in a

clear matrix. The underlying tubule base-

ment membrane

is

comprised of an outer

layer of fine filaments and an inner gran-

ular zone. Connective tissues and base-

ment membranes of insects are frequently

multi-layered (e.g. Ashhurst, '68; Locke

and Huie, '72; Oschman and Berridge,

'70) but the functional significance of the

various components is poorly understood.

W e

suspect that the collagen fibers are

necessary to provide a n elastic protective

sheath around the highly contractile tu-

bules, and that the basement membrane

may be comprised of polyelectrolytes that

can act both as a mechanical filter and

as a charged sieve to restrict the move-

ment of certain molecules into or out of

the basal infoldings (e.g. Oschman and

Berridge, '7

1 .

The basal infoldings form deep chan-

nels extending perpendicular to the base-

ment membrane. These chan nels probably

correspond to the fine cytoplasmic str ia-

tions in the same region observed by light

microscopists (Snodgrass, '35, p. 418). The

infoldings are long channels of narrow

but uniform width that extend deep into

the basal cytoplasm of the cells. Although

the channels appear empty in convention-

ally prepared specimens (fig. 7), when

specimens are fixed in glutaraldehyde-lan-

thanum or in glutaraldehyde-calcium fol-

lowed by

in

block

treatment with uranium-

calcium (fig. 8) both the channels and the

basement membrane are dense. There

are two interpretations of this finding:

some of the lanthanum or uranium is in

a colloidal form which simply becomes

trapped in the channels and thus acts as

an extracellular marker much like col-

loidal lan tha num; alternatively, fixing and

processing with lanthanum or calcium-

containing solutions may precipitate and

retain some negatively charged extracel-

lular substance. Further study is needed

to clarify this finding.

Figures 7 and

9

reveal that the ba-

sal channels are not formed from simple

folding of the basal plasma membrane

but instead ar e developed from interdig-

itating finger-like extensions of neigh-

boring cells. A similar arrangement oc-

curs in vertebrate kidney and salivary

gland striated duct and is well illustrated

in 3-dimensional reconstructions published

by Rhodin

( 58),

Tandler ('62), and Bulger

('65). Th at these a re indeed processes from

adjacent cells is documented in figure 9,

which

is

a tangential section through the

basal surfa ce in a region where one of

the cells is more darkly stained than the

other. Comparison with figure

7

shows

that the infolds actually arise because of

irregular arborizing extensions from ad-

jacent cells. Toward the basal surface

these extensions often contain microtu-

bules, which are sectioned transversely in

figure 7 and longitudinally in figure 9.

The microtubules are often within

1

of

the basement membrane and parallel to

the direction of the extensions. The mi-

crotubules may have a role in the genesis

and maintenance of the basal labyrinth,

as will be discussed below. No specialized

structures such as slit diaphragms have

been observed a t the openings of the basal

infolds.

In addition to the microtubules men-

tioned above, the cytoplasm within the

interdigitating processes from adjacent

cells contains mitochondria , ribosomes, and

segments of smooth and rough endoplas-

mic reticulum (fig. 7). In some regions

one encounters chain s of smooth vesicles

and tubules (fig. lo). In surface view (e.g.

top of fig. 25) these structures form a n

extensive reticular network. A similar ar-

rangement occurs in the ciliary epitheli-

um of the vertebrate eye (Tormey, '64) in

which the reticular structure is known to

be an artifact of fixation caused by the

disruption and vesiculation of a flattened

smooth surface cisternal system. Because

of the similarity of this struc ture in the

Malpighian tubule with that of ciliary epi-

thelium, we a re unsure of its exact struc-

ture and think that the chains of vesicles

Fig. 9

Tangential section n ear surface, middle

region. One cell is lighter t han its neighbor, em-

phasizing int erdig itatin g processes. Microtubules

shown in

transverse section

in

figures 6 and

7

occur he re in lo ngitu dinal profile (arrows). oriented

parallel to the sides

of

the interdigitating proc-

esses. In upper

left

basement membrane compo-

nen ts are observed with collagen and other fibrils

oriented parallel to surface as in figure 6 . x 40,000.


11/42

M A L P I G H IA N T U B U L E S T R U C T U R E A N D F U N C T I O N 275


12/42

276

B . J . WALL. J .

L.

O S C H M A N AND B . A. SCHMIDT

Fig.

10

Association

of

mitochondria with membranes

of

interdigitating processes

from

adjacent

cells.

Apparent ch ains

of

vesicles within t he interdigitations may be

sheets

of smooth

endoplasmic reticulum that have vesiculated during fixation.

X 40,500.

could be artifacts caused by distortion

of

a smooth surfaced cisternal system.

Cytoplasmic inclusions

The conspicuous and distinguishing fea-

ture

of

the middle region is the presence

of large vacuoles. While many of these

appear to be empty in sectioned material,

others contain either a network of fine

filaments, fine granules, or large concre-

tions apparently comprised

of

concentric

shells of densely staining material (figs.

5, 1 1 ,

25).

Although the latter are usually

within the cytoplasm (fig. ll , one was

found within a nucleus (fig. 12). These

structures are common in Malpighian tu-

bules as well as in insect intestinal cells

(Gouranton, 68), insect utricles (Ballan-


13/42


277

Fig. 11

Membranebound vacuole containing mineral concretion found

within

cytoplasm

of

middle

region.

X

40,500.

DufranCais, 70), protozoa (Andre and

Faure-Fremiet, '67), and many other in-

vertebrates. The nature of these concre-

tions has been a matter of controversy.

Earlier studies (Berkaloff, '58, '59; Sri-

vastava, '62; Fuller, '66) suggested that

these structures contained urates. Wig-

glesworth and Salpeter ('62) noted that

the intracellular refractile concretions

were not the same as the excretory gran-

ules

in

the lumen, as only the latter gave

a positive urate test with ammoniacal sil-

ver nitrate. Gouranton ('68) was unable to

detect uric acid or guanine by chromatog-

raphy

of

intestinal concretions. Instead,

he detected calcium, magnesium, iron,

phosphate, carbonate, protein, and acid

mucopolysaccharide. Similarly, Mello and

Bozzo ('69) tentatively recognized lipopro-

tein or phospholipid in excretory globules

in Malpighian tubules of larval bees. Stad-

houders and Jacobs ('61) did histochemi-

cal studies on the structures in cockroach-

es and found they contained calcium and

phosphorus. We thus suspect that the con-

centric concretions of Periplaneta tubules

are sites of calcium phosphate storage.

They are probably not uric acid since:

1) uric acid is not a major excretory

product in cockroaches (Mullins and Coch-

ran, '72); (2) urate crystals observed in

electron micrographs of cornea of gout

patients (Slansky and Kuwabara, '68) show

a cuboidal crystalline structure that dif-

fers considerably from the concentric con-

cretions in the Malpighian tubules; and


14/42

278

B . J . W A L L , J .

L .

O S C H M A N A N D

B.

A . S C H M I D T

Fig.

12

Mineral concretion within

a

nucleus,

middle region.

X

9.000.

( 3 )

the structures in Malpighian tubules

resemble closely the calcified concretions

(lithosomes) in protozoa (Andre and

Faure-Fremiet,

67)

and in experimentally

induced calcification of mammalian kid-

ney tubules (Giacomelli et al., 64). The

concretions are occasionally found in the

tips of microvilli (fig. 2 0 , and may enter

the lumen in this way.

In addition to mitochondria, ribosomes,

and endoplasmic reticulum, one occasion-

ally encounters structures resembling an-

nulate lamellae (fig. 13). This figure also

illustrates a dense body that is similar to

that shown in figure

3.

The functional

significance of these inclusions is unclear.

However, we suspect that they may be

analogous to the microbodies or peroxi-

somes of vertebrate tissues. Microbodies

typically have a crystalline core, as do at

least some of the dense granules in Mal-

pighian tubules (fig. 14). In many species

microbodies participate in the metabolism

of ammonia and uric acid, and the possi-

ble role of these organelles in nitrogen

excretion will be considered in the D I S -

In addition to the spherical vacuoles

described above, some sections of the tu-

bules contain clear vacuoles with a more

prismatic shape (fig. 15). These structures

could contain either a substance with low

intrinsic electron opacity, or their content

may be extracted at some stage of proc-

essing. If urates are stored in these Mal-

pighian tubules, it is more likely that they

would be within prismatic granules such

as these than in the larger refractile con-

cretions. We suggest this because the

urate-containing crystals in insects usu-

ally have needle, lens, or lozenge shapes

(Noel and Tahir,

29)

similar

to

many of

the inclusions

in

figure 15. Further, Wig-

glesworth

(53)

reported that the uratic

granules in

Rhodnius

tubules are soluble

in aqueous fixative and are rapidly dis-

solved by osmium. Figure 15 also illus-

trates an autolysosome, Golgi complex, and

multivesicular body.

The mitochondria in the middle region

lack the crystalline inclusions encoun-

tered in the distal tubule. However, in

one specimen we have observed a region

in which most of the mitochondria were

irregular or doughnut shaped, contain-

ing central clear regions of density and

texture similar to the cytoplasm (figs.

16,

17). In some cases (fig. 17) the mitochon-

drial cristae are organized in a spoke-like

fashion around the central core. We have

only observed these mitochondria in one

specimen, and are unable to determine

their signficance.

Other inclusions are arrays of tubular

structures (fig. 18) that are sometimes

associated with granular material (fig. 19)

that

is

not bounded by a membrane. The

nature of these structures is unknown,

but we have observed similar inclusions

in nearby tracheal cells, indicating that

they may be due to a non-specific patho-

logical condition such as a virus.

Apical

surface

The apical cell surface is folded into

microvilli of the same sort a s those found

in

the distal region. However, the degree

to which mitochondria penetrate into the

microvilli seems to be less than in the dis-

tal region (compare figs. 2 and

5 )

and in

CUSSION.


15/42


2

79

Fig.

1 3

Annulate lam ellae (al) and vacuoles containing dense granular m aterial (d). Th e

latter may be precursors of the large concentric concretions such a s illustrated in figures 1 1

and 12. or they ma y be microbodies or peroxisomes, as shown in figure 1 4 .

x

80.000.

Fig. 1 4 Dense body w ith crystalline core (arro w) similar to that found in vertebrate

peroxisomes. X

49,000.

some areas , no mitochondria are found same cells. Note tha t in figure

5

the micro-

within microvilli (figs.

16,

20,

21).

There villi

in

the cell shown in profile at the

is

variation

in

the exten t of penetration bottom and the cell shown at the top have

of mitochondria into microvilli within the areas where no mitochondria are within


16/42

280 B . J. WALL,

J.

L. O S C H M A N A N D B . A . S C H M I D T

Fig.

15

Second type

of

inclusion

(::')

observed within cells

of

middle region.

Low

density

of

these prismatic structures suggests that their crystalline contents were dissolved during

fixation. Autolysosome

A ) ,

Golgi complex

(G)

and multivesicular body

(MVB). X 22,000.

microvilli and other areas that have many. extensions of the network of smooth en-

In the areas where mitochondria are ab- doplasmic reticulum that is abundant

in

sent, there are still two sorts

of

microvilli, the apical zone of cytoplasm interior to

those penetrated by smooth ER and small- the microvilli (fig. 20). We are uncertain

er ones, lacking inclusions

fig. 21).

The

if

this smooth endoplasmic reticulum is

tubules within the microvilli are clearly continuous with that extending into the


17/42


281

Fig. 16 Atypical mitoch ondr ia, mid dle region of tubule. These mitochondria conta in on e

or more pockets

of

cytoplasm

( ) .

Their functional significance is unknown. Also included i s

a profile

of

Golgi complex (G).

X

21,000.

interdigitating processes at the basal cell

surface (figs.

6,

7, 10).

Cell junctions

At the interface between adjacent cells

we have noted two sorts

of

specialized junc-

tions. The most extensive in terms of area

of contact is the septate junction, which

is characterized

by

a series of thin dia-

phragms or septa extending perpendicular

to the plasma membrane (fig. 23). Ura-

nium-calcium treatment (fig. 22) renders

the spaces between septa opaque to elec-

trons.

so

that the 3-dimensional structure


18/42

282

B.

J .

WALL,

3. L.

OSCHMAN AND B.

A .

SCHMIDT

Fig. 18 Cells of middle region sometimes con-

tain early stages of autophagy (st ructu res bounded

by isolation m emb ranes , im ) and later stages (lyso-

Fig.

17

Higher magnification

of

atypical mi- some,

1).

Included

is

a t.s.

of

a crystalline array

of

tochondria containing pockets

of

cytoplasm. Note tubules (t) of unknown significance. Golgi com-

spoke-like configuration

of

mitochondrion at top, plex (G) and endoplasmic reticulum ar e also in-

and m ultivesicular body (mvb). X

75,000.

cluded. X 29,000.


19/42

M A L P I G H I A N TUBULE S T R U C T U R E A N D F U N C T I O N

283

Fig. 19 Basal surface, middle region. There is some evidence of pinocytosis (arrow) but

this is not frequently observed in this tissue. Cells contain gra nular inclusions (*), on e of which

is surrounded by tub ular struc tures similar to those illustrated in ficure 18. X 41 000.

of the septa is seen

in

relief as parallel ignated as comb desmosomes by Danilova

folded sheets of intermembranous mate- et al.

('69).

Images similar to those

in

rial. Junctions

of

this sort were first de- figure

22

are obtained with lanthanum

scribed by Locke

('65)

and have been des- staining, and have been integrated with


20/42

284

B .

J .

WALL, J . L.

OSCHMAN

AND B . A . SCHMIDT

Fig. 21 Transverse section of microvilli, mid-

dle region. Profiles of smooth endoplasmic reticu-

lum ar e circular in tran sverse sections. Smaller

microvilli lack mitochondria or smooth endoplas-

mic reticulum. X 66,000.

freeze fracture data (e.g. Flower, '70) to

provide a structural model for this junc-

tion (Gilula et al.,

'70).

Gap junctions are

also encountered occasionally, and, again,

they have a characteristic

appearance

n

face views of material treated with ura-

nium-calcium

in

block. This type

of

image

has been obtained previously

n

vertebrate

tissues with lanthanum infiltration (Revel

and Karnovsky,

'67).

Again, freeze etch

evidence has been utilized to ascertain a

possible 3-dimensional reconstruction of

the junctional structure (McNutt and

Weinstein, '70).

Symbiotic pro tozoa

In some of the specimens we have ob-

served symbiont protozoa within the proxi-

mal portion of the middle region. Figure

Fig.

20

Apical surface, middle region. Micro-

28

illustrates the ultrastructural appear-

ance of these symbionts, while their

illi contain tubular structures that ar e continu-

ous with smooth endoplasmic reticulum (arrow).

Note dense concretion

hat

appears to

be pinch. tion within the tubule is illustrated in

ing

off

into

lumen

(L). X

40,000.

figure 27. These symbionts are similar


21/42


285

Fig.

22

Face view of sept ate junc tion after glu-

taraldehyde-calcium fixation and uranium acetate-

calcium

in block

stain ing. Urani um fills extracellu-

lar space delineating septa as pleated sheets.

X 18,000.

Fig.

23

Septate junct ion, middle region.

X

65,000.

in structure to the haplosporidians de-

scribed by Woolever ('66) in the cockroach,

Leucophaea .

These protozoa attach to the

tubule cells by means of microvilli tha t

insert between the microvilli of the tubule

cells. These sporozoa produce spores wi th

a thick cuticle that is difficult to section.

Fig.

24

Stellate cells. Note in figure b that

A

detailed description

of

the protozoa

is

stellate cell is comp arabl e in size to nucle us (n p)

not

appropriate in

this paper, although we

o f

primary cell. Photomicrographs

of

middle

re-

gion fixed in Carnoy's fixative and stained with

will discuss below their possible involve- aqueous toluidine blue. Stellate cells have acquired

ment in excretion. a blue color. x 1,250.


22/42

286

B . J. W A L L . J . L. O S C H M A N

AND B .

A . S C H M I D T

Fig.

25

Basal portion, ste llate cell. Note ab sence

of

vacuoles and presence

of

numerous

multivesicular

bodies.

Stellate cell nucleus (N).

X 21,000.


23/42

M A L P I G H I A N T U B U L E S T R U C T U R E A N D FUNCTION

87

Fig. 26

Stellate cell in region whe re it exten ds across whole tubule. Organelles are less

abundant than

in

prim ary cells. and m icrovilli lack inclu sion s such a s mitochondria or smooth

endoplasmic reticu lum . Basal interdigitations ar e sim ilar to those of primary cells.

X

11,500.

Stellate

cells

Whole-mounts stained with toluidine

blue show that the tubules contain a sec-

ond smaller sort

of

cell that occurs at

regular intervals along the length of the

tubule (fig. 24). A comparable second cell

type has been noted in a number of pre-

vious studies (reviewed by Taylor, '71b),

but their function remains obscure. In the

cockroach the stellate cells have smaller

nuclei than primary cells, and have nar-

row processes that extend between adja-

cent primary cells. Because of their small-

er size they present narrow profiles in

transverse sections and are encountered

infrequently in electron micrographs. The

stellate cells are characterized by the ab-

sence of vacuoles or concretions

so

abun-

dant in primary cells (figs.

25,

26). The

basal surface

of

stellate cells

is

elaborated


24/42

288 B. J.

WALL,

J . L.

OSCHMAN AND

B. A .

SCHMIDT

into long processes that interdigitate with

those of the primary cells (fig. 25). The

cytoplasm often appears less dense than

that of primary cells, apparently because

of fewer free ribosomes (fig. 9 probably

shows the interdigita tion between a pri-

mary cell and a lightly stained stellate

cell). There are numerous multivesicular

bodies, lysosomes, segments of rough en-

doplasmic reticulum, and Golgi complexes

with associated small vesicles (figs. 25,

26). The mitochondria are of similar struc-

ture to those in primary cells, although

they seem to be less abundant (fig. 26).

The apical surface is folded into micro-

villi, but these differ from those in pri-

mary cells in that they do not contain

extensions of smooth tubular endoplasmic

reticulum or mitochondria (fig. 26).

Proximal region

We have designated a s the proximal

region the short portion of the tubule

(about 0.5 mm long) that drains into the

ampulla . Th e transition in morphology of

the tubule cells at the junction between

middle and proximal regions is apparent

in light micrographs of methylene blue-

stained thick sections (fig. 27). The proxi-

mal region does not contain symbionts

and there

is

an abrupt change in the form

of the brush border. Electron micrographs

(figs. 29, 30) reveal that cells of the prox-

imal region have microvilli that are thin-

ner and farther apart than those

of

middle

and distal regions. It will be seen below

that microvilli in the proximal region

closely resemble those within the ampulla

and midgut. For purposes of comparison

the reader is referred to other studies that

illustrate aspects of insect midgut struc-

ture (Oschman et al., '74; Berridge, '70;

Oschman and Wall, '72; Smith et al., '69).

Another feature in common with midgut

is that the basal channels are somewhat

distended to form a compar tment of rela-

tively large volume that opens to the hemo-

coel in relatively few places (fig. 30; see

Berridge, '70). Finally there

is

an inclu-

sion in these cells that

is

also found in

the ampulla. This is a dense membrane-

bound accumulation of membrane-like

leaflets with a wavy appearance in sec-

tions. This inclusion

is

illustrated in the

proximal tubule (figs. 29, 30) and in the

ampulla (fig. 32).

Fig. 27

Photomicrog raph of

1

p thick methyl-

ene blue-stained section of region where tubule

drains into ampulla A) . Symbionts (S) are pres-

ent in middle region. Note abrupt transition in

cell density an d in b rush border at junctio n be-

tween middle and proximal regions (arrow). X 820.

The connective tissue and musculature

investing the tubules is most fully devel-

oped in the proximal region (figs. 29,

30).

The cells lack the vacuoles and concre-

tions a bundant in the middle region. How-

ever, the proximal portion of the middle

region also lacks vacuoles (e.g. fig. 28).

In general, the mitochondria within the

proximal tubule cells seem more concen-

trated toward the apical or luminal sur-

face, rather than scattered throughout the

cells a s in middle and dist al regions of the

Fig. 28 Section

of

middle region tubule near

proximal en d. Protozoan sym bionts (s) (haplospo-

ridia) a re in lum en. Thin processes from th e pro-

tozoan cells mingle with microvilli

of

Malpighian

tubule. M uscle ( m ) is well developed in th is re-

gion.

x

12,200.


25/42

MALPIGHIAN

TUBULE STRUCTURE AND FUNCTION

289


26/42

290

B . J . WALL J. L . OSCHMAN AND B.

A .

SCHMIDT

Fig. 29

Survey view, proximal region of tubule. Lumen

is

triangular in shape, basal sur-

face is heavily invested w ith connective tissue an d mu scula ture

(m).

Microvilli ar e fur the r

apart than in other regions. Basal infolds are distend ed. Dense bodies (arro ws) have lam ellar

interiors similar to those in am pul la cells (figs. 32, 34). X 4,100.

tubules (figs. 2,

5).

The cytoplasm also nor smooth tubules extend into the micro-

contains lysosomes, rough endoplasmic

re-

villi. Instead, each microvillus has a core

ticulum, Golgi complexes, and numerous of fine filaments similar to those within

multivesicular bodies. There is generally midgut microvilli (e.g. Smith et al.,

'69).

a region at the cell apex that is free of Cells

of

the proximal region interdigitate

organelles except for small vesicles and extensively along both basa l and latera l

filaments (fig.

30).

Neither mitochondria borders.


27/42


291

Fig. 30 Proximal region a t higher m agnification. Connective tissue (ct) is thicker th an

basemen t mem brane (bm) a nd ha s muscles (m ) embedded in it . Cells interdigitate, but basal

cha nne ls (bc) between a djace nt cell processes ar e wider tha n in middle region (compar e with

figs. 7,

19).

Microvilli ar e no t closely pack ed and r esemb le thos e of am pu lla r cells (figs.

32,

33):

Dense lamellate inclusions (arrow) ar e similar to those found in amp ullae

(fig. 3 2 ) . X 14.000.

Ampulla

interdigitations similar to those of the prox-

Figure

31

summarizes the organization imal region (figs. 29, 30). As mentioned

of

the ampulla. The lumen of the

proxi-

above, the ampulla contains large mem-

ma1 region narrows at the region where brane-enclosed structures (about 2-3

p

in

it

drains into the ampulla

(figs.

27, 31). diameter) with a laminated interior (figs.

The epithelium lining the ampulla is col- 32, 34). Although we do not know the

umnar and has thin microvilli

and

basal function

of

these structures, they resem-


28/42

292

midgut

B . J . W A L L , J.

L.

OSCHMAN AND B .

A .

SCHMIDT

-Middle

leum

Fig. 31 Sum mary diag ram of ampu lla a nd associated structures. Muscles are not in-

cluded. Proximal part of middle region contains protozoa (fig.

27).

Proximal region of tubu le

is composed of cells similar to ampulla. Tubule lum en narrows at region where it d rains into

ampu lla. Cavity of am pulla d ra in s via nar row slit into gut. Apical surfaces of tubules, am -

pullae, an d midgut ar e folded to form microvilli. Ileum is lined with cuticle.

ble the osmiophilic lamellate bodies found

within atrial and parabronchial cells of

vertebrate lung (Lambson and Cohn,

'68;

Hatasa and Nakamura, '65; Smith and

Ryan,

73).

The dense bodies in the lung

are formed in the rough endoplasmic re-

ticulum and, when released to the cell

surface, contribute to the surface active

agent (surfactant) that coats the alveolar

membrane. Although we have not studied

the fate of the dense bodies in the insect

ampulla, they also may be released at the

cell surface, possibly contributing to the

surface coating of the drainage canals

(see below) or hindgut.

Another striking feature of the ampulla

is the presence of a deeply penetrating

canalicular system. Sections through these

canaliculi (e.g. fig.

34)

give the appear-

ance of clear vacuoles lined with a surface

that has some rudimentary microvilli pro-

jecting inward and containing a small

amount of membranous and granular ma-

terial. These structures can be observed

in methylene blue-stained thick sections.

By tracing these structures in serial sec-

tions, we have found that they are deeply

penetrating channels that approach close-

ly the apical cell surfaces. The canaliculi

do

not seem to open into the ampullar

lumen, but we are not entirely certain

of

this point. Fig. 27

is

one of a series of sec-

tions, and shows a region where one cana-

liculus approaches the ampullar lumen

near the region where a proximal tubule

drains. We have no information on the

function of this system, but suspect that

it could provide a stationary or unstirred


29/42

MALPIGHIAN TUBULE

STRUCTURE

AND FUNCTION

293

Fig.

33

Transverse section

of

microvilli of am-

pulla. Th ese microvilli a re irregular i n sh ap e and

are not closely packed as in tubules (compa re with

figs. 4, 21).

x 66 000.

fluid compartment from which solutes

could be reabsorbed by the ampullar cells.

We are not aware that such structures

have been described in other transporting

epithelia, although we have observed some-

thing resembling them but on a smaller

scale in cockroach midgut (unpublished).

Clearly, this part

of

the excretory system

should be studied further.

D rainage c ana l

Figure

31

illustrates the region where

fluid from the ampulla drains into the gut.

We have not been able to work out the

precise structure of this region, which ap-

pears to consist

of

a complex labyrinth

of channels. The individual cells lining

the channels (fig.

35)

are thin and highly

Fig. 32 Survey view of ampulla. Only a por-

tion of thick connective tiss ue is inc lud ed. Cells

conta in peculiar lamellated inclu sion s (arrows). Mi-

tochondria are particularly abu nda nt near apical

surface.

X 5,000.


30/42

294

B . J .

WALL,

J.

L. OSCHMAN AND B . A . SCHMIDT

Fig. 34 Cavities within ampullar cells. These

clear spaces seem to be bounded by apical me m-

brane, since short microvilli protrude i nto them.

They do not appear to be permanently open to

the ampullar lumen. They contain membranous

fragments (arrows) and particulate material that

renders their contents somewhat more dense than

the lumen (compare with

fig.

32). X 5,000.

folded on their apical but no t their basal

surfaces. Canaliculi are present. The mi-

crovilli resemble those of the midgut even

more closely than do those

of

the ampulla.

The cells contain lipid droplets, small mi-

tochondria, numerous ribosomes, and oc-

casional bits of rough endoplasmic reticu-

lum. The basal surface faces the gut and

appears to have little if any basement

membrane. The apical surface faces the

drainage canal and is coated with a loose

layer of material that looks like a dis-

organized jumble of membranes resem-

bling a peritrophic membrane . Although

direct evidence is lacking, we suspect that

this layer could be formed by release of

the contents

of

the dense lamellated bodies

in the ampulla.

IZeum

We have mentioned the morphological

resemblance between cells of the ampulla

and those of the midgut, and referred for

comparison to the num erous published de-

scriptions of midgut structure. To empha-

size this point, we include a brief descrip-

tion

of

the beginning of the hindgut,' the

ileum. The ileum of

Periplane ta

has not

been described elsewhere although Ballan-

Dufrancais ('72) has described Blatte l la

ileum. The ileum

is

lined by a thin cutic-

ular intima secreted by columnar cells.

The basal surface is irregular (fig. 36)

and there is a layer

of

connective tissue.

Mitochondria are abundant only toward

the apical pole of the cells. At low mag-

nifications (fig. 36) one observes dense

bands of material lying adjacent to the

lateral plasma membranes. Higher mag-

nification (fig. 37) reveals that these are

bundles of microtubules. The apical plas-

ma membrane is elaborated into closely

packed tubular infolds (figs.

36, 37).

The

cuticle consists of a dense epicuticle and

a pale endocuticle. In some areas the en-

docuticle contains clear oval or lens-shaped

inclusions that either arise from or give

rise to clear prismatic crystals

in

the api-

cal cytoplasm of the cells (fig. 37). Very

little information is available on the phys-

iology of the ileum, and further ana-

tomical description would be pointless at

present.

DISCUSSION

M e c h a n i s m

of

secre t ion

The striking anatomical feature of Mal-


31/42

MALPIGHlAN

T U B U L E

S T R U C T U R E

AND F U N C T I O N

295

Fig.

35

Cells bordering drainage canal from ampulla. Canal lumen at bottom, midgut

lumen at top. Cells are thin and have microvilli resembling those

of

midgut. Canaliculi x c )

are abundant. Cells contain rough endoplasmic reticulum (rer), mitochondria (m), and lipid

droplets (L). Loose surface coat ( sc ) resembles a peritrophic membrane.

x 30,000.

pighian tubules revealed

by

this and pre- merular kidney, proximal and distal kid-

vious ultrastructural studies is the pres- ney tubules, etc. (Pease, '56; Fawcett, '62;

ence

of

highly folded cell surfaces.

This

Berridge and Oschman, '72). All

of

these

feature is characteristic of many other se-

epithelia secrete a fluid that is about is-

cretory tissues such as pancreas, sweat osmotic with blood and that is nearly pro-

and salivary glands, choroid plexus, aglo- tein-free (except where protein secretion


32/42


33/42

MALPIGHIAN TUBULE STRUCTURE

AND

FUNCTION

297

Fig. 37 Higher magnification, apical portion of ileum. Cytoplasm con tain s bundle s of

microtubules (mt). Lumen is a t bottom. Apical mem bra ne is highly folded. Clear crystalli ne

inclusions ( ) with rectang ular profiles occur wi thin cytoplasm an d cuticle. Those ne ar outer-

most surface of the cuticle a re more rounded in profile. x 30,000.


34/42

298 B .

J.

W A L L ,

J.

L . O S C H M A N A N D

B . A .

SCHMIDT

flux, the osmolality of the transported fluid

is proportional to the osmolality of the

bathing fluid, addition of non-permanent

solutes to the bathing medium brings

about a proportional increase in the con-

centration of the transported ion, and os

molality of the transported fluid is not

dependent on rate of fluid flow (Oschman

and Berridge, 71). A s mentioned in the

beginning of the article, a local osmotic

gradient model has been proposed to ac-

count for fluid transport in these diverse

epithelia. Solute uptake from basal infolds

of Malpighian tubules is thought to gener-

ate the osmotic driving force that causes

water to be taken up from the hemolymph

into the tubule cells. Solute pumping into

spaces between microvilli may provide the

local or standing osmotic gradient that

draws water from the cell into the lumen

(Berridge and Oschman,

69).

Apparently

potassium pumps are involved in estab-

lishing the gradients in Malpighian tu-

bules of many insect species, since the

secreted fluid usually contains a high con-

centration of potassium (Ramsay,

53;

Ber-

ridge, 68).

Figure

8

compares the transporting

Malpighian

tubule

38

Fig. 38 Comparison of gallbladder and Malpighian tubule ultrastructure, approximately

to the scale indicat ed. Gallbladder produces isosmotic fluid reabsorption and h as long narr ow

intercellular spaces that are folded into thin leaflets. Malpighian tubule secretes a nearly

isosmotic fluid secretion, an d has num erou s closely packed microvilli. Gallbladder structur e

based on micrographs of Kaye et al.

(66)

and Tormey and Diamond (67). Malpighian

tubule dimensions based on present study.


35/42


299

cells of gall bladder and Malpighian tubule

at about the same scale. Each Malpighian

tubule cell has thousands of microvilli,

while a gall bladder cell has only a single

intercellular space. Since both of these

cells transport water in isosmotic propor-

tions, it is implied that the thousands of

short microvillar and basal channels of

the Malpighian tubule cell may be func-

tionally equivalent to a single intercellular

space of gall bladder. While the contribu-

tion of each microvillar channel is prob-

ably quite small (Taylor, 71a) the effect

of many such small gradients, when

summed over the entire cell surface, may

be comparable to that of the intercellular

spaces of gall bladder.

Taylor (71a) has presented an alternate

simple osmotic model in which the cells

are hyperosmotic to the hemolymph and

the lumen hyperosmotic to the cytoplasm.

This model might seem to apply well to

Periplaneta

Malpighian tubules, since the

secreted fluid is hyperosmotic to the bath-

ing medium (table 1). However, the tubu-

lar fluid in many insects is isosmotic or

even hypoosmotic to the hemolymph (Ram-

say, 54; Maddrell, 71). Indeed, i t was the

failure of such simple osmotic theories

to account for the general phenomenon

of isosmotic fluid secretion that led to the

development of the local osmosis concept

(e.g. Auricchio and Biirany, 59).

Another explanation of fluid secretion

is the formed body hypothesis of Riegel

(70), summarized in figure 39. Formed

bodies are lysosome-like spherical vesicles

20

p or more in diameter that are ob-

served in micropuncture samples obtained

from various transporting tissues (reviewed

by Riegel, 70). The formed bodies are

thought

to

contain protein and proteases.

Riegel suggests that the formed bodies

are secreted into the lumen of the Mal-

pighian tubule or other transporting tissue

(fig. 39a). The proteases become activated,

resulting in the hydrolysis of the proteins

(fig. 39b). This increases the osmotic pres-

sure within the formed bodies, water en-

ters them by osmosis, and the formed bod-

ies swell (fig. 39c). Solutes within the

lumen that are unable to penetrate into

formed bodies are concentrated as water

is drawn into the formed bodies (fig. 39d).

The increased osmotic pressure of the lu-

minal fluid then draws water across the

39

Fig. 39 Formed body hypothesis for fluid se-

cretion (Riegel,

70):

(a ) Formed body is secreted

from cell into lume n. (b) Digestive enzy mes within

formed body hydrolyse proteins. ( c ) Increase in

solute concentration within formed body causes

HzO to enter by osmosis, and formed body swells.

(d) Lumen becomes filled with swollen formed

bodies. S olutes un ab le to enter formed body be-

come concentrated in lumen . (e) Water is draw n

into lumen by osmosis.

f)

Hydrostatic pressure

increases within lumen d ue to water entry, and

fluid flows through lum en.

epithelial cell layer by osmosis (fig. 39e).

While Riegel

(68,

70)

presents an assort-

ment of evidence supporting this hypoth-

esis, formed bodies fail to explain a num-

ber of the important characterist ics of fluid

secretion by Malpighian tubules and other

transporting epithelia. For example, if

formed bodies are responsible for fluid se-

cretion, why is there such a strict depen-

dence of fluid secretion on ion secretion

(e.g. Oschman and Berridge, 71)? Absence

of transportable solutes in the bathing

medium brings about immediate cessation

of transport in gall bladder, intestine, Mal-

pighian tubules, and other transporting

systems. Long term secretion by the formed

body mechanism would require a contin-

uous

synthesis of polypeptides. However,

Malpighian tubules isolated from Calli-

phora can secrete in a simple medium

containing a single substrate such as mal-

tose, pyruvate, or an individual amino acid

(Berridge, 66). This result, together with

the finding that inhibitors of oxidative

phosphorylation stop fluid secretion (Ber-

ridge, 66) indicate that fluid secretion

depends on ion transport driven by ATP

hydrolysis, rather than by the alternate


36/42

300

B .

J .

W A L L ,

J .

L . O S C H M A N AND

B . A . SCHMIDT

synthesis and breakdown of polypeptides,

However, the formed bodies should not be

ignored, and further study may reveal that

they have a role in the excretory process.

Finally, Wessing and Eichelberg ('75)

have arrived a t a n entirely different model

of secretion based on extensive histochem-

ical studies of Drosophila Malpighian tu-

bules. These workers have suggested that

ions are transported across the tubules in

association with mucosubstances, a model

similar to that envisioned by Philpott ('68)

for the chloride cells of teleost fish.

G e n e s i s a n d m a i n t e n a n c e of c h a n n e l

ge om e t ry .

An aspect of secretion that

ha s been neglected

is

the manner in which

highly folded cell surfaces are formed dur-

ing

development, and how they are main-

tained during secretion, when there are

local changes in hydrostatic pressure pro-

duced by water flow. In the Malpighian

tubules we find microtubules within the

basal inte rdigitations. Microtubules are

known to be involved in the formation

and maintenance of cell shape (de-The,

'64; Branson, '68; Byers and Porter, '64;

Gibbins et al., '69; Tilney and Gibbins,

'69), and their position within the basal

interdigitations is consistent with a simi-

lar role

in

the Malpighian tubule. If it is

correct that fluid is actively absorbed from

the basal channels, they would then tend

to collapse. The extracellular mater ial that

is apparently stained with lanthanum and

uranium (figs. 7, 8) could be involved in

maintaining the channel in the optimal

sha pe for fluid absorption.

Role of t h e a m p u l l a . Observations on

freshly dissected cockroaches show that

the ampullae accumulate the fluid se-

creted by the tubules and then, when full,

contract vigorously to force that fluid into

the intestine. This arrangement has two

consequences. First, the tubular fluid is

forced through the labyrinthine drainage

canal, which may act as a one-way valve

preventing back-diffusion from the gu t into

the ampulla. Secondly, the tubular fluid

remains in contact with the cells lining

the ampulla during the interval between

ampullar contractions. The possibility thus

arises that the tubular fluid may be modi-

fied while in the ampulla. Ultrastructural

evidence supports

an

absorptive role for

the ampulla. since its cells resemble those

gan of nutrient absorption in the insect.

Particularly striking are similarities in

configuration of basal interdigitations,

brush border microvilli, and concentra-

tion of mitochondria toward the apical

surface. Although one cannot be certain,

these structural similarities are at least

suggestive of common functional charac-

teristics. The tubular fluid contains amino

acids, sugars, and other substances that

are essential and therefore must be re-

absorbed (Ramsay,

58;

Farquha rson, '74;

Maddrell and Gardiner, '74). Little is

known

of

the location or mechanism of

sugar and amino acid absorption, although

Balshin and Phillips ('71) reported active

reabsorption of amino acids by the rectum.

The hindgut of Sarc ophuga larvae absorbs

bicarbonate ions in exchange for amm onia

(Prusch, '71). In the cockroach the colon

fluid i s usually hypoosmotic to the primary

urine and hemolymph (Wall, '70) suggest-

ing so lute reabsorption in this portion of

the gut. The present study indicates that

the ampullae and possibly the proximal

part

of

the Malpighian tubules can be re-

garded anatomically as extensions of the

gut, and hence may be the first sites of sol-

ute reabsorption from the primary urine.

This could be of particular advantage dur-

ing antidiuresis, as it would allow more

complete reabsorption of essential metab-

olites from the tubular fluid when the

rate of urine formation is probably slow.

The large vacuoles or canaliculi in the

ampulla (fig. 34) may be involved in re-

absorption.

There have been other suggestions that

the proximal regions in some tubules are

reabsorptive (e.g. Patton and Craig, '39;

Srivastava, '62; Wigglesworth, '31c). The

proximal region of

Calpode s

Malpighian

tubules reabsorbs ions (Irvine, '69) while

the comparable portion of

R h o d n i u s

tu-

bules reabsorbs potassium and water (Wig-

glesworth, 31c).

Finally, the ampullae of cockroaches are

not structurally analogous to the pylorus-

bladder (the first part of the hindgut) of

Core thra larvae (Schaller, '49) since the

pylorus-bladder is not a diverticulum but

a segment of the gut. However, the py-

lorus-bladder contracts rhythmically at a

rate that

is

regulated by certain neuro-

hormones (Gersch, '67) and the same may

of the hidgut, which

is

the principal or-

be true of the ampulla.'


37/42


30 1

Em bry oge ne s i s of a m p u l l a e a n d

M a l p i g h ia n t u b u l e s

The close ultrastructural resemblance

between the cells of proximal tubule, am-

pulla, and midgut has a bearing on the

embryological origin of the Malpighian tu-

bules, a controversial topic in the past.

Opinion has differed on whether the tu-

bules are derived from midgut or hindgut

(reviewed by Snodgrass, 35; Srivastava

and Khare, '66). The hindgut or procto-

daeum is thought to be entirely ectoder-

mal, although Henson ( 32) suggested that

the anterior part of the proctodael invag-

ination is endodermal in origin. The mid-

gut or ventriculus, on the other hand,

arises by regeneration of the mesenteron

rudiments, cells from the endodermal cell

layer surrounding the archenteron. In most

insects the Malpighian tubules seem to

have a histological resemblance to the mid-

gut, and their endodermal origin has been

supported by Tirelli ('29), Savage ('56,

'62), and Butt ('49). Srivastava and Khare

('62) disagree with this view, since the

Malpighian tubules in

Phi losamia

(Lepi-

doptera) bud off from the blind end of

the proctodaeal invagination before the

mesenteron has formed.

In the present study we have found that

the histological resemblance between the

Malpighian tubules and midgut is even

more striking at the ultrastructural level.

The proximal tubules have similar micro-

villi and basal infoldings, and are ana-

tomically continuous with the ampulla and

midgut. Srivastava and Khare ('62) point

out that histological similarity does not

prove common embryological origin. How-

ever, the ultrastructural and probable

functional similarities between the proxi-

mal region of the tubule, ampulla, and

midgut imply that these tissues are de-

rived from endoderm. There is an abrupt

change in the morphology of the cells at

the junction of the proximal and middle

regions of the tubules, leaving us uncer-

tain as to the origin of the middle and

distal regions of the tubule. Henson ('32)

has suggested that tubules originating

from the midgut can become secondarily

attached to the hindgut. It is conceivable

that the reverse could occur, i.e., the tu-

bules could arise from the anterior end

of the proctodaeum and later become at-

tached to the midgut. Such migrations

of developing tubules seem improbable

however, and clearly do not occur in

Blat ta

or iental i s and Periplane ta in which the

tubules grow out from the ampulla (Hen-

son, '44; Schmidt, unpublished).

Ex c re t ion he ro l e

of

pe rox i som e s

It has been generally thought that in-

sects are uricotelic. Ammonia is toxic and

therefore has been considered suitable as

an excretory product only in aquatic forms

and in a few terrestrial invertebrates (re-

viewed by Campbell, 73). Although many

insects do excrete uric acid, at least dur-

ing a part of their life cycle, ammonia

has been found to be a major excretory

product in the cockroach, Periplane ta

am e r i c ana

(Mullins and Cochran,

72,

'73a,b). Ammonia accounted for up to 91

of the total excretory nitrogen, depending

on diet. Uric acid was not detected in fecal

extracts, even in animals fed high protein

diets. Little uric acid was excreted even

when i t was fed to the animals. Instead,

much of the uric acid was absorbed and

stored in the fat body.

Cockroach Malpighian tubules contain

structures resembling mammalian micro-

bodies or peroxisomes (fig. 14) and these

structures may be involved in nitrogen

excretion. Peroxisomes have common func-

tional properties in organisms as diverse

as T e t r a h y m e n a yeasts, beans, and man

(DeDuve, '69). Peroxisomes contain a num-

ber of enzymes related to nitrogen metab-

olism, including various L and D-amino

acid oxidases, uricase, or urate oxidase,

and catalase (reviewed by Hruban and

Rechcigl, '69). The amino acid oxidases

catalize the direct formation of ammonia

from amino acids. Likewise, peroxisomes

are involved in the metabolism of uric

acid in many species (Shnitka, '66) ac-

cording to the scheme:

uricase

uric acid 2 H 2 0

llantoin COn 2 HzOz

catalase

2

H202

H20 2

Hydrogen peroxide is formed and rapidly

decomposed by catalase, which apparent-

ly is universally present in peroxisomes.

These organelles have also been observed

in

Call iphora

Malpighian tubules (Berridge

and Oschman, '69) as well as in other

insect tissues (Locke, '69; Locke and

Mc-

Mahon, '71) but their precise role in ni-


38/42

302

B.

J. WALL. J. L. OSCHMAN AND B . A .

SCHMIDT

trogen metabolism has not been estab-

lished in insects.

Ex c re t ion he ro le of syrnbionts

The symbionts observed within the mid-

dle region of the tubules of

Periplane ta

resemble the haplosporidians found in

Bla t t e l l a ge nnan ic a .

Woolever ('66) has

made

a

remarkably thorough study of the

latter. She found that the

Blatte l la

sym-

bionts enter the host orally, multiply with-

in the Malpighian tubule cells, migrate

into the tubule lumen where they under-

go schizogony and sporogony, and release

their spores into the gut. The close ultra-

structural resemblance between vegeta-

tive and spore stages in

Blatte l la

and

Peri-

p lane ta

indicates that the

Periplane ta

sym-

biont could also be a haplosporidian.

Little is known about the metabolic

in-

teractions between the tubule symbionts

and their hosts. The symbionts ar e located

at the proximal end of the middle region

of the tubule (figs. 27, 28). They are not

present in the proximal region of the tu-

bule, possibly because the symbiont micro-

villi are adapted for attachment only to

the closely packed brush border of the

middle region. Since the primary urine

resembles a filtrate of the hemolymph,

the symbionts are bathed in a constantly

renewed medium that

is

rich in nutrients.

In addition they can readily release their

spores into the digestive tract so they will

be extruded with the feces.

Dr. K.

G .

Purohit has pointed out to us

that the symbionts might benefit their

hosts in m uch the same manner as do

those or ruminants (cows, sheep, etc.).

The rumen symbionts synthesize proteins

from amino acids and ammonia. These

proteins become available to the host when

the symbionts pass on into the intestine

and

are

digested. The symbionts also pro-

duce volatile fatty acids that are absorbed

directly across the rumen epithelium (Hun-

gate,

'68;

Dobson and Phillipson, '68).

The protozoan symbionts

in

the Malpighi-

an tubules produce spores which are not

attacked by digestive enzymes in the gut

(Woolever, '66). However, the vegetative

cell disintegrates when the spores are re-

leased. Fragments of the vegetative cells

thus pass into the gut and

are

digested

(Woolever, '66), possibly providing an ad-

ditional source of nutrients. The symbionts

may be able to synthesize essential amino

acids that the cockroach is not able to

manufacture d e

nouo. A

symbiotic rela-

tionship of this sor t has already been dem-

onstrated between

Blatte l la

and the bac-

terial symbionts within the mycetocytes

of the fat body.

Blat te l la

that lack these

symbionts a re smaller and less fecund than

infected roaches (Donnellan and Kilby,

'67), are unable to synthesize six amino

acids that normal animals can (Henry and

Block, '60, '62), and have heavier deposits

of urates in their fat body (Brooks and

Richards,

'56;

Pierre, '62). The bacterial

symbionts can be isolated from the host

and grown on a medium in which uric

acid is the only carbon source and can

degrade the uric acid to pyruvate or am-

monia (Donnellan and Kilby, '67). Com-

parable studies done on

Periplaneta

have

indicated that the fat body symbionts

are

responsible for the synthesis of folic, ascor-

bic, and pantothenic acids (Gallagher, '63).

Thus the symbionts within the Malpighian

tubules could have an important role in

metabolism in the insect.

ACKNOWLEDGMENTS

This study was begun in the laboratories

of Professors Michael Locke and Bodil

Schmidt-Nielsen at Case Western Reserve

University, Cleveland, Ohio, with support

from National Institutes of Health Grants

AM09975 and GM09960. The research

was also supported by USPHS Fellowship

GM24015 to

B.

J.

Wall and

AM

29555 to

J.

L. Oschman, as well as National Insti-

tutes of Health grants FR-7028 and

AM

14993. We are indebted to our colleagues

who have provided us with advice and

support.

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